We are studying the molecular mechanisms that control the differentiation of thymic epithelial cells (TECs), which are required to promote T-cell development within the thymus. Initial TEC differentiation depends on the function of Foxn1, which encodes a forkhead transcription factor gene, providing a unique resource to investigate Foxn1 function. Foxn1 homozygous null mice (nude mice) are functionally athymic, arresting TEC differentiation at a very early stage. Using gene targeting we have generated a new allele for Foxn1, denoted Foxn1/delta. This allele encodes a truncated protein in which the DNA binding and activation domains are intact, but a portion of the N-terminal domain is deleted. Adult Foxn1delta/delta mice have small thymi with specific defects in thymocyte development, including a 40-fold drop in thymocyte number and specific defects in thymocyte development. Foxn1delta/nu mice, with one delta and one null allele, have even more severe defects. These defects are similar to those seen in mice with mutations in Kit and IL-7 signaling, and RT-PCR for kit ligand (kit-I) showed a near total absence of kit-I message in Foxn1delta/delta thymus. Surprisingly, the Foxn1delta/delta and Foxn1delta/nu phenotypes are thymus-specific, with apparently normal skin and hair development, identifying a thymus-specific activity for the Foxn1 N-terminus, which previously had no known function. Foxn1delta/delta, Foxn1delta/nu, and Foxn1nu/nu therefore constitute an allelic series for the Foxn1 gene, providing a unique resource to investigate Foxn1 function. We propose 4 specific aims to allelic series for the Foxn1 gene, providing a unique resource to investigate Foxn1 function. We propose 4 specific aims to test the hypothesis that the Foxn1delta mutation encodes a hypomorphic allele that allows normal initiation of TEC differentiation and thymus organogenesis, but disrupts a thymus-specific functional domain required for subsequent TEC differentiation. test the hypothesis that the Foxn1? mutation encodes a hypomorphic allele that allows normal initiation of TEC differentiation and thymus organogenesis, but disrupts a thymus-specific functional domain required for subsequent TEC differentiation. Our aims are :1) to determine the earliest stage at which thymus development is affected in Foxn1delta/delta and Foxn1delta/nu embryos; 2) to identify the specific thymocyte and TEC defects during fetal and post-natal development in Foxn1delta/delta and Foxn1delta/nu mice; 3) to test whether the Kit-l andIL7 signaling pathways act downstream of Foxn1; and 4) to determine whether the Foxn1 N-terminal domain acts as a thymus-specific protein-protein interaction domain. Through these approaches, we will for the first time be able to study the role of Foxn1 in TEC development and function beyond the initial stages of thymus organogenesis. Further, our identification of a new function for the N-terminal domain may have implications for functional domains in other forkhead family transcription factors.